Identification of small molecule agonists of the orphan nuclear receptors liver receptor homolog-1 and steroidogenic factor-1

Article abstract of DOI:10.1021/jm060990k

We report the identification of substituted cis-bicyclo[3.3.0]-oct-2-enes as small molecule agonists of subfamily V orphan nuclear receptors (NR5A), liver receptor homolog-1 (LRH-1) and steroidogenic factor-1 (SF-1). Using fluorescence resonance energy transfer (FRET)-based biochemical assays, compound 5a (GSK8470) was identified as a high-affinity ligand for LRH-1 and SF-1. In liver cells, 5a increased the expression of the LRH-1 target gene small heterodimer partner (SHP). Synthesis of analogues modified at three positions led to the development of compounds with functional selectivity between LRH-1 and SF-1.

Full text of DOI:10.1021/jm060990k

6652
J. Med. Chem. 2006, 49, 6652-6655
Unlike most NRs, LRH-1 and SF-1 bind to DNA as
monomers and show constitutive activation of transcription when
expressed in cells.3,4 Receptor activity can be regulated by
phosphorylation, sumoylation, or through interaction with the
atypical orphan receptors SHP (NR0B1)12 and DAX-1 (NR0B2)13
that lack their own DNA-binding domains (DAX-1 ) dosage
sensitive sex reversal - adrenal hypoplasia congenita gene on
the X chromosome, gene 1). Recently, X-ray crystallography
coupled with mass spectroscopy has revealed the presence of
phospholipids in the ligand binding pockets of both human
LRH-1 and SF-1.14-17 The functional role of phospholipids in
the regulation of receptor activity remains to be established and
is further confounded by the absence of a lipid in the pocket of
the mouse LRH-1.18 However, the knowledge that human
LRH-1 and SF-1 can bind to phospholipids suggests that the
receptors are chemically tractable and has further stimulated
interest in their potential role as therapeutic targets for drug
discovery. The identification of a synthetic activating ligand
would be a valuable chemical tool to elucidate their function
in mammalian physiology.2
Identification of Small Molecule Agonists of the
Orphan Nuclear Receptors Liver Receptor
Homolog-1 and Steroidogenic Factor-1
Richard J. Whitby,† Sally Dixon,† Patrick R. Maloney,‡
Philippe Delerive,| Bryan J. Goodwin,‡ Derek J. Parks,‡ and
Timothy M. Willson*,‡
School of Chemistry, UniVersity of Southampton, Southampton,
HANTS, SO17 1BJ, U.K., Molecular DiscoVery Research,
GlaxoSmithKline, 5 Moore DriVe, Research Triangle Park,
NC 27709-3398, and CardioVascular & Urogenital Center of
Excellence for Drug DiscoVery, GlaxoSmithKline,
25 AVenue du Quebec, 91951 Les Ulis, France
ReceiVed August 16, 2006
Abstract: We report the identification of substituted cis-bicyclo[3.3.0]-
oct-2-enes as small molecule agonists of subfamily V orphan nuclear
receptors (NR5A), liver receptor homolog-1 (LRH-1) and steroidogenic
factor-1 (SF-1). Using fluorescence resonance energy transfer (FRET)-
based biochemical assays, compound 5a (GSK8470) was identified as
a high-affinity ligand for LRH-1 and SF-1. In liver cells, 5a increased
the expression of the LRH-1 target gene small heterodimer partner
(SHP). Synthesis of analogues modified at three positions led to the
development of compounds with functional selectivity between LRH-1
and SF-1.
A high throughput screen using a fluorescence resonance
energy transfer (FRET)-based assay to detect the interaction of
the ligand binding domain of human LRH-1 with a peptide
derived from the coactivator TIF2 (amino acids 737-757) led
to the identification of the unusual bicyclic compound 5a19 as
a potent ligand with EC50 ) 430 nM [TIF2 ) transcriptional
intermediary factor 2 (NCOA2)]. It also proved to be an
effective ligand for human SF-1 with EC50 ) 54 nM in a FRET-
based assay for recruitment of a peptide derived from DAX-1
(amino acids 1-23). In both assays, the apo-receptors were
initially present with 1 equiv of phospholipid bound, and it is
remarkable that the small nonpolar molecule 5a was able to
displace these large endogenous lipids from their pockets. The
potent binding affinity of 5a and rigid cis-bicyclo[3.3.0]oct-2-
ene core structure made the series an attractive template for
structure-activity studies. As well as establishing the require-
ments for high-affinity binding, we were particularly keen to
discover compounds with selectivity for LRH-1 over SF-1 and
vice versa. Compounds were designed to define the ligand-
receptor pharmacophore at three positions (R1-R3 in Table 1).
Compound 5a was produced in a one-pot tandem reaction
sequence in which initial co-cyclization of a 1,6-enyne 1 using
the Negishi reagent (Cp2ZrBu2)20 to afford a zirconacyclopen-
tene 2 was followed by insertion of phenyl isonitrile to generate
the potent carbometalating reagent, zirconocene η2-imine 3
(Scheme 1). Subsequent insertion of 4-octyne into the carbon-
zirconium bond followed by protonolysis gave the bicyclic
amine 4a. Unexpectedly, 4a underwent a facile rearrangement
to afford the 1-amino-cis-bicyclo[3.3.0]oct-2-ene compound 5a
as revealed by a small molecule X-ray structure determination.19
Variation of the starting enyne 1 allowed the synthesis of a series
of analogues 5b-5g with modification in the R1 group (Table
1). Two methods were used to accomplish the rearrangement
of the initial products 4 to the required bridgehead amines 5.
Initially, for compounds 5a-5c, 5h, and 5j, we relied on the
spontaneous rearrangement that occurred partly during the initial
workup with methanol/water and completed during chromatog-
raphy on silica (method A). A drawback was that the product
5 decomposes on silica, making the yields and initial purity of
the products capricious. Subsequently, for compounds 5d-5g,
5i, and 5k we isolated the initial amine product 4 by protonation
Nuclear receptors (NRs)a are a large family of mammalian
transcription factors that function as the molecular targets for
widely prescribed medicines and therapeutic agents in clinical
development.1 The activity of many NRs is controlled by small
lipophilic ligands such as steroid hormones, retinoids, vitamin
D, and thyroid hormone. On the other hand, a multitude of NRs
for which the natural ligands are unknown have been termed
“orphan” receptors.2 We have a particular interest in the liver
receptor homolog-1 (LRH-1)3 and steroidogenic factor-1 (SF-
1),4 members of the subfamily V nuclear receptors (NR5A) for
which no small molecule ligands have been reported.
LRH-1 plays a critical role in embryonic development of the
endoderm.5 In adults it is expressed in the intestine, liver,
exocrine pancreas, and ovary. LRH-1 regulates the expression
of genes involved in hepatic bile acid biosynthesis and
cholesterol homeostasis.3 Thus, the receptor may be a target
for the treatment of cardiovascular disease. Its regulation of
aromatase expression also suggests a possible utility in cancer
therapy.6,7 SF-1 plays an important role in sex determination
during development. SF-1 knockout mice were phenotypically
female independent of genetic sex.8 The receptor is expressed
in the testes, ovaries, and adrenal cortex where it regulates many
genes involved in steroid hormone production.4,9 It is also
expressed in the hypothalamus and has been implicated in the
regulation of feeding behavior.10,11
* Address correspondence to Timothy M. Willson, Discovery Medicinal
Chemistry, GlaxoSmithKline, 5 Moore Drive, NTH-M1421, Research
Triangle Park, NC 27709-3398. Tel: (919) 483-9875. Fax: (919) 315-
0430. E-mail: tim.m.willson@gsk.com.
† University of Southampton.
‡ Molecular Discovery Research, GlaxoSmithKline.
| Cardiovascular & Urogenital Center of Excellence for Drug Discovery,
GlaxoSmithKline.
a Abbreviations: DAX-1, dosage sensitive sex reversal - adrenal hypo-
plasia congenita gene on the X chromosome, gene 1; FRET, fluorescence
resonance energy transfer; LRH-1, liver receptor homolog-1; NR, nuclear
receptor; SHP, small heterodimer partner; SF-1, steroidogenic factor-1; TIF2,
transcriptional intermediary factor 2 (NCOA2).
10.1021/jm060990k CCC: $33.50 © 2006 American Chemical Society
Published on Web 10/20/2006

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23 6653
Table 1. Synthesis and Biological Activity of 1-Arylamino-cis-bicyclo[3.3.0]oct-2-enes
LRH-1c
SF-1c
(SD
yield
EC50
(µM)
REd
(%)
EC50
(µM)
REd
(%)
cmpd
methoda
(%)b
R1
R2
R3
(SD
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
A
A
A
B
B
B
B
A
B
A
B
C
D
D
C
D
D
C
D
D
D
D
C
53
61
68
53
19
27
35
34
49
7
42
58
29
27
39
34
29
20
13
30
16
28
14
Ph
Me
n-C4H9
c-C6H11
4-Br-C6H4
3-MeO-C6H4
2-naphthyl
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
(E)-4-oct-4-enyl
(E)-4-oct-4-enyl
(E)-4-oct-4-enyl
(E)-4-oct-4-enyl
(E)-4-oct-4-enyl
(E)-4-oct-4-enyl
(E)-4-oct-4-enyl
(E)-3-hex-3-enyl
(E)-6-dodec-6-enyl
CH(Me)C4H9
CH(Me)C8H17
Me
n-C6H13
c-C6H11
n-C12H25
Ph
n-C6H13
n-C6H13
n-C6H13
n-C6H13
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
0.43
0.09
0.022
0.012
0.16
0.10
0.06
1.4
0.07
0.04
0.007
0.002
0.01
0.01
0.01
0.2
100
15
50
50
70
115
55
50
0.054
>10
0.008
50
0.030
0.010
0.17
0.14
0.13
0.8
0.014
0.13
0.13
0.001
0.002
0.07
0.01
0.05
0.1
0.003
0.05
0.04
95
90
35
45
30
80
30
90
95
>10
0.07
0.32
2.1
0.034
0.23
0.01
0.05
0.7
0.003
0.02
45
25
20
45
25
>10
0.043
0.18
0.008
0.07
100
65
>10
>10
0.15
0.33
1.0
0.02
0.004
0.7
35
45
15
30
0.12
0.56
0.7
0.02
0.02
0.3
55
70
25
20
3-F-C6H4
4-Cl-C6H4
2,3-diMe-C6H4
4-OCF3-C6H4
4-tert-Bu-C6H4
4-I-C6H4
Ph
Ph
Ph
Ph
Ph
Ph
0.03
0.01
0.011
0.008
5t
>10
>10
>10
>10
5u
5v
5w
n-C6H13
n-C6H13
n-C6H13
>10
>10
>10
1-naphthyl
1.2
0.3
45
a See text and Scheme 1 for description of methods A-D. b Based on enyne 1 for methods A and B, and on cyclopentenone 6 for method C and D. c All
biological data n ) 3. d RE, relative efficacy normalized to 5a for LRH-1 and 5m for SF-1, (5%.
Scheme 1a
Subsequent acid-catalyzed displacement of the hydroxyl moiety
with aniline gave the desired amines 5l-5p (Table 1) in variable
yields (methods C and D). Although the tertiary alcohols 7 could
be obtained pure, their instability toward acid meant that higher
overall yields often resulted if they were used crude (method
D). The exceptionally facile acid-catalyzed displacement of the
hydroxyl with an amine is probably driven by release of strain
energy from the starting bicyclo[3.3.0]oct-1-ene. It was incon-
venient to use different isonitriles to allow variation in the group
R3 in the zirconium route, so we chose to employ a range of
anilines to displace the alcohol in compound 7 (R2 ) n-hexyl)
to afford the amines 5q-5w (Table 1).
The compounds 5a-5w were screened for activity against
both LRH-1 and SF-1 using FRET-based peptide recruitment
assays, and the results are shown in Table 1. The data are
presented as an EC50, which serves as a measure of the binding
affinity for the receptor, and the relative efficacy at peptide
recruitment, which in the absence of a known standard was
normalized to 5a for LRH-1 and 5m for SF-1. Several trends
were discerned in the data. Changing R1 from aryl to a similar
size alkyl group increased the binding to LRH-1 but lowered
the relative efficacy, as seen in the comparison of 5a with 5c
and 5d. For example, compound 5d (EC50 ) 12 nM) is around
40 times more potent than 5a, although efficacy is reduced by
half. On SF-1, a different structure-activity relationship was
observed since 5c and 5d showed only a small increase potency
compared to 5a, but a large increase in relative efficacy. Larger
aryl groups (5e-5g) gave mixed results on both receptors,
suggesting a limit to the size of the lipophilic pocket. Compound
5f, with a 3-methoxy substituent on the aryl ring, is interesting
as it slightly increases both binding affinity and efficacy on
LRH-1 relative to 5a. A methyl group at either R1 or R2 (e.g.,
5b and 5l) yielded compounds with low efficacy on LRH-1 and
no measurable activity on SF-1. A larger branched alkenyl group
at R2 was tolerated in SF-1 but not in LRH-1, resulting in the
identification of the functionally selective analogue 5i. Other
alkyl, cycloalkyl, and phenyl substituents at R2 (e.g., 5m-5p)
showed parallel changes in activity. Increase in the size of R3
a
Reagents and conditions: (a) Cp2ZrBu2, -78 °C to 20 °C, then 20
°C, 2 h; (b) PhNC (0.95 equiv) -35 °C to 20 °C; (c) RC≡CR Or
RCHdCH2, 20 to 60 °C, 2 h; (d) MeOH/H2O or aq. NaHCO3; (e) Method
a: MeOH, then SiO2 or Method B: aq. NaHCO3 then PhNH2 (10 equiv),
camphorsulfonic acid (0.1 equiv) 20 or 50 °C, 2 h; (f) Co2(CO)8, DMSO
(6 equiv), THF, 50 °C, 4.5 h; (g) R2MgBr, CeCl3; (h) R3NH2 (10 equiv),
camphor sulfonic acid (0.1 equiv), 20 °C, 16 h or 65 °C, 2-6 h.
of the zirconacycle under basic conditions (aqueous NaHCO3)
and treated the crude product with 10 equiv of aniline and 0.1
equiv of camphor sulfonic acid to effect the rearrangement to
5. Purification of the final product was accomplished by
chromatography on basic alumina (Method B). Generally, the
yields of the latter method were lower than the former but less
variable.
Variation of the substituent R2 was initially accomplished by
using different alkyne and alkene traps for the intermediate η2-
imine complex 3 (Scheme 1) to provide compounds 5h-5k
(Table 1). An alternative route to the same cis-bicyclo[3.3.0]-
oct-2-ene structures was developed based on Pauson-Khand
cyclization21,22 of enyne 1 to afford the bicyclic cyclopentenone
6. The cerium chloride promoted23 addition of various Grignard
reagents (R2MgX) gave the tertiary alcohols 7 in good yield.

6654 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23
Letters
Supporting Information Available: Synthetic methods and
analytical data for compounds in Table 1, and biological methods
for the Table 1 and Figure 1. This material is available free of
charge via the Internet at http://pubs.acs.org.
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stability and oral activity are underway.

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23 6655
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JM060990K